In the Colorimetric Protease Assay Kit (Cat. No. 23263), my sample absorbance values are lower than smallest trypsin or other protease standard values. Why?
It could be that the sample protease activity is low relative to the range of dilutions used for the standard curve. You can prepare and use additional standard dilutions to ensure that the full range of assay sensitivity is utilized. Alternatively, the sample protease activity is low relative to the protease used to make standard curve. You can prepare a standard curve using the known amounts of the specific protease being tested.
In the Colorimetric Protease Assay Kit (Cat. No. 23263), all of my sample absorbance values for non-trypsin proteases are low. What can I do?
There are two potential causes for low absorbance values in the unknown samples. The first being that pH and other buffer conditions were not optimal for the protease being tested. You can repeat the assay using buffer conditions that are optimal for the protease being tested and used to make standard curve. Secondly, it is possible that the protease being tested digests the casein substrate more slowly or less fully than the trypsin standard for which the default assay method was developed. You can perform the digestion step for >20 minutes (e.g., 40 mins); if necessary, also perform the TNBSA development step for longer. You can also use the specific substrate for the protease being tested.